FR3065214A1 - MATRIX FOR THE SPECIFIC DETECTION OF ALKALOIDS BY MALDI-TOF MASS SPECTROMETRY - Google Patents

Abstract

The present invention relates to a novel bithiophenic matrix for the specific detection of alkaloids by MALDI-TOF mass spectrometry.

Description

DESCRIPTION

Technical area

The present invention relates to (i) a new compound used as a matrix in a method for analyzing molecules of low mass which may be less than 800 Da, in particular alkaloids, by mass spectrometry (MS) of the MALDI-TOF type, said method being generally designated under the acronym MALDI-TOF-MS for method of analysis by mass spectrometry (Mass Spectrometry) with time-of-flight with matrix-assisted laser desorption-ionization or MALDI (Matrix Assisted Laser Desorption Ionization), (ii) the method of manufacturing said compound, (iii) its use as a matrix in MALDI-TOF-MS, and (iv) said method of analysis in MALDI-TOF-MS using said compound as a matrix.

The present invention finds in particular an application in the pharmacological field.

In the description below, the references in square brackets ([]) refer to the list of references presented at the end of the text.

State of the art

Alkaloids represent a class of interesting secondary plant metabolites with a wide range of biological activities. Lewis bases, they are generally the subject of a long and expensive extraction process, exploiting their differential solubilities according to the pH of the medium and leading to a crude extract designated under the term of alkaloid totum (AT). Having been used in traditional medicine for a very long time, alkaloids are also valuable compounds in modern pharmaceuticals although many of them such as aconitine or strychnine are also known to have serious toxic effects [1-3] .

For these reasons, methods facilitating rapid and reliable detection of alkaloids are essential for many applications in clinical and forensic toxicology [4], as well as for the quality control of food supplements and pharmaceutical products [4-8]. Providing maximum information, mass spectrometry (MS) and in particular electrospray ionization (ESI) has become a standard technique for the analysis of small molecules including alkaloids. ESI is generally linked with liquid chromatography (LC), thus providing a more powerful tool for the analysis of complex mixtures. However, LC-MS requires extensive sample preparation and experimental optimization procedures to obtain the best results. In addition, the use of non-volatile buffers, which is often necessary to achieve sufficient separation of the alkaloids, limits the applications of LC-MS.

Like MALDI, ESI represents a soft ionization technique, which is commonly applied to the analysis of large molecules such as peptides, proteins or oligosaccharides. However, compared to ESI, MALDI or MALDI-TOF type mass spectrometry requires practically no sample preparation and provides almost instant results. In addition, unlike ESI, which produces only quasi-molecular ions (M + H) ⁺ , the MALDI or MALDI-TOF technique produces both radical (M) ' ⁺ and quasimolecular (M + H) ions. ⁺ [9, 10]. This considerably widens the range of directly detectable compounds [11], Since the simplicity and the time necessary for the development of a method are important factors for routine analysis, the MALDI technique could therefore provide a useful complement to the approaches. LC-MS classics for the analysis of small molecules, typically with a molecular mass of less than 800 Da, such as alkaloids.

However in mass spectrometry of the MALDI or MALDI-TOF type, if the matrix plays an essential role during the desorption / ionization process, it also represents a limiting factor of the technique since it generates very often and in large quantities of “parasitic” ions of the matrix (matrix noise) in the range of low mass-to-charge ratios (m / z). This phenomenon thus makes the characterization of small molecules, typically <800

Da, very delicate or even impossible, especially if the latter are only present in small quantities. Matrix peaks can indeed mask those of the analytes in the region of interest (ie 0 <m / z <800) (Figure 1) [12], Alkaloids, a major class of compounds of pharmacological interest, are not immune to this rule.

Strategies to minimize this effect include lowering the ionization energy, while respecting certain matrix / analyte mixture ratios [13], but also the addition of agents that inhibit matrix noise [14-16], En furthermore, the study of the formation of matrix noises could help to choose the suitable matrix for a given analytical problem. In addition, some arrays can generate very few signals even when irradiated at high laser energy.

In addition to presenting a low matrix noise, the interaction with the analytes to be tested is certainly the most important factor characterizing a "good" matrix. Recently Cramer has classified MALDI matrices into two generations: small molecules which have been empirically discovered as MALDI matrices and those which have been created by structural modifications of these compounds in order to improve or refine their adapted physicochemical properties. to the MALDI phenomenon [17], some first generations of matrices have been shown to have affinities for certain chemical families, but their ionization profile has never been exclusively selective for a specific group. On the other hand, recently designed second generation matrices often exhibit markedly improved selectivity, as is the case for the detection of oligosaccharides by 6-hydrazinonicotinic acid [18]. Recently 3- [5 '(methylthio) -2,2'-bithiophen-5-ylthio] propanenitrile (or MT3P) was presented as a new matrix for the selective ionization of alkaloids in MALDI-TOF type mass spectrometry [19]. However, the performance of MT3P limits its field of application, in particular in terms of toxicological analyzes.

There is therefore a real need for a matrix which overcomes the drawbacks and obstacles of the prior art, in particular a method making it possible to specifically analyze small molecules <800 Da, such as, for example, alkaloids, in particular to characterize and / or quantify them from a complex mixture.

Description of the invention

The present invention relates to the design of a new matrix, PFPT3P, generating few “parasitic” ions on the one hand and, due to privileged π-π interactions between the latter and the nitrogen heterocycles, leading to other part in the specific ionization of small molecules with molecular mass less than 800 Da, in particular alkaloids. It then becomes possible to characterize and quantify, including from complex mixtures (raw plant extracts, biological fluids, etc.), these molecules with molecular mass less than 800 Da of very great pharmacological interest by MALDI-TOF- MS.

Thus, on the basis of the model compound MT3P, the inventors evaluated five new compounds of bithiophenic matrix for their matrix properties with respect to five selected alkaloids, biologically very active and / or toxic such as colchicine, sparteine, pilocarine , harmony and strychnine. In addition, the selectivity of ionization has been tested on compounds belonging to different chemical families such as curcuminoids, benzopyrones, flavonoids, steroids and peptides. All these bithiophenic matrices have been compared with two of the most commonly used commercial matrices: α-cyano-4hydroxycinnamic acids (CHCA) and 2,5-dihydroxybenzoic acids (DHB), as reference compounds. In addition, matrix noise formation was evaluated by calculating the scores for matrix suppression effects (MSE) for all the compounds tested [13],

Based on these results, the most promising candidate appeared to be the compound PFPT3P which showed the best ionization of the alkaloids, superior to DHB although less strong than CHCA when tested on pure compounds. PFPT3P was then tested on complex mixtures such as a crude extract of Colchicum autumnale (colchicum, Colchicaceae), the RYTMOPASC® solution [pharmaceutical tincture based on broom (Cytisus scoparius, Fabaceae) containing sparteine] , and human plasma enriched in strychnine. The performance of PFPT3P was then found to be much higher than that of MT3P while retaining the selectivity obtained with the latter, and while no ionization of the target compounds in the RYTMOPASC® solution and the plasma enriched in strychnine was obtained with CHCA.

The simple synthesis of PFPT3P is carried out in two stages with a yield of 75%, from commercial products and at low cost. PFPT3P constitutes a new matrix which, in MALDI-TOF-MS type spectrometry, significantly improves the quality of the spectra, generates very few parasitic ions, has excellent selectivity with respect to the alkaloids which it makes it possible to detect. , including in a complex biological medium such as plasma with very low detection (LOD) or quantification (LOQ) limits, without any special preliminary preparation. This matrix of PFPT3P therefore offers a great potential for numerous applications in the case of (semi) quantitative analyzes. Depending on the quality of the abacuses obtained, it is indeed possible to develop specific assay methods for alkaloids exploiting the very high sensitivity of MALDI-TOF type mass spectrometers leading to detection (LOD) or quantification (LOQ) limits very low.

The present invention therefore relates to the use of the PFPT3P compound of formula : F F. Jx F he = F © 1 F as a matrix in PFPT3P Exact mass: 462.96 a mass spectrometry device with

desorption / ionization under laser assisted by a matrix or MALDI.

According to a particular embodiment of the present invention, said compound is used to analyze qualitatively and quantitatively a molecule of molecular mass less than 800 Da or a mixture of molecules or a sample of the crude organic extract or biological liquid type, said mixture or said sample comprising at least one molecule of molecular mass less than 800 Da, said molecule of molecular mass less than 800 Da being able to be chosen from alkaloids.

By "raw organic extract" within the meaning of the present invention means any extract obtained from fresh or dry materials from living beings such as, for example, Archaea, Bacteria, protists, Mushrooms, Plants, Animals and n ' having undergone any subsequent purification step. We distinguish in particular, raw plant extracts.

By “biological liquid” within the meaning of the present invention, is meant all the liquids derived from living beings of human or animal origin. Mention may be made, for example, of blood, plasma, fluids of the genital mucosa, seminal fluid, semen, cervical mucus, fluids of the skin such as sweat, fluids of dander, effusion fluids or closed cavities, liquids from the digestive or urinary system.

By “alkaloids” within the meaning of the present invention is meant basic nitrogenous heterocyclic organic molecules which may have pharmacological activity. Generally, alkaloids are derivatives of amino acids. Biosynthesized alkaloids are found as secondary metabolites, mainly in plants, fungi and a few small animal groups. In addition to the alkaloids of natural origin, there are also synthetic analogues, manufactured by total synthesis such as chloroquine, chloroquinine or by hemisynthesis as in the case of vindesine or vinorelbine. Finally, substances of an alkaloidal nature may not have a natural origin, for example the case of pyrazole derivatives. Although many alkaloids are toxic (like strychnine or aconitine), some are used in medicine for, for example, their analgesic properties (like morphine, codeine), as part of sedation protocols (anesthesia ) often accompanied by hypnotic properties, or as an antimalarial agent (quinine, chloroquinine) or anticancer agent (Vinblastine, vincristine). The alkaloids are categorized according to their chemical structure. We distinguish:

• Group of Azolidines (pyrrolidines): Aniracetam, Anisomycin, CX614, Dextromoramide, Diphenyl prolinol, domoic acid, Histapyrrodine, kainic acid, Methdilazine, Oxaceprol, Prolintane, Pyrrobutamine, hygrine, cuscohygrine.

• Group of Azines: Piperidine, Conicine, Trigonelline, Arécaidine, Guvacine, Pilocarpine, Cytisine, Nicotine, Sparteine, Pelletierine.

• Tropane group: Atropine, Hyoscyamine, Cocaine, Ecgonine, Scopolamine.

• Quinoline group: Acridine, Bicinchoninic acid, Broxyquinoline, Chlorquinaldol, Cinchophen, Clioquinol, Dequalinium, Dihydroquinine, Dihydroquinidine, Hydroxychloroquine, 8-Hydroxyquinoline, lodoquinol, Kynurenic acid, Mefloquine, Quininine, Prininine, Prininine , xanthurenic acid, Strychnine, Brucine, Veratrine, Cevadine, Echinopsine;

o Aminoquinolines: Chloroquine, Hydroxychloroquine, Primaquine; o 8-Aminoquinolines: Tafenoquine, Rhodoquine, Pamaquine.

• Isoquinolines group: Dimethisoquine, Quinapril, Quinapirilat, Debrisoquine, 2,2'-Hexadecaméthylènediisoquinolinium dichloride, Nlaurylisoquinolinium bromide, Narcéine, Hydrastine, Berbérine;

o Opium alkaloids:

Natural: Morphine, Codeine, Thebaine, Papaverine, Narcotine, Noscapine; Semi-synthetic: Hydromorphone, Hydrocodone, Heroin;

Synthetics: Fentanyl, Pethidine, Methadone, Propoxyphene.

• Group of Indoles:

o Ergolines: The ergot alkaloids (Ergometrine, Ergotamine, Ergosine, Ergovaline, Ergokryptine, Ergocornine, Ergocristine, Lysergic acid, etc.), Ergine, LSD ...

o Beta-carbolines: Harmine, Yohimbine, Réserpine, Emétine.

• Terpenoids group:

o The alkaloids of aconite napel: Aconitine; o Solanidine, Solasodine, Batrachotoxine, Delphinine;

o Steroids: Solanine, Samandarin.

• Betaine group (quaternary ammonium compounds, they are not alkaloids in the literal sense, even if regularly classified as such): Muscarine, Choline, Neurine.

• Pyrazole groups.

The molecule (s) with a molecular mass of less than 800 Da and / or the alkaloid (s) defined above can thus be detected and quantified in samples of biological liquids or of raw organic extract, in particular of raw plant extract, without require preliminary purification or separation steps by mass spectrometry under laser assisted by matrix.

According to a particular embodiment of the present invention, the sample is chosen from crude organic extracts, or any fraction obtained from said crude organic extracts, and biological liquids, of human or animal origin.

A subject of the present invention is also the PFPT3P compound of formula:

PFPT3P

Exact mass: 462.96

The present invention also relates to the use of the PFPT3P compound of formula:

F. F .F yes c _X S ^ CN F ' 1 F to make a matrix PFPT3P Exact mass: 462.96 intended for use in a

mass spectrometry with desorption / ionization under laser assisted by matrix or MALDI.

The present invention also relates to a method of manufacturing a matrix intended to be used in a mass spectrometry device with desorption / ionization under laser assisted by matrix or MALDI comprising the or consisting in the crystallization of the PFPT3P compound of formula;

PFPT3P

Exact mass: 462.96

The MALDI device can be a mass spectrometer coupling a matrix-assisted laser ionization source and a time-of-flight analyzer.

According to a particular embodiment of the process for manufacturing said matrix according to the present invention, said crystallized PFPT3P compound is then optionally dissolved in an organic solvent or water comprising a sample to be analyzed or directly dissolved in the sample to be analyzed, said solvent or water then being, if necessary, vaporized, ultimately forming a crystallized matrix of PFPT3P compound comprising the sample to be analyzed.

By “organic solvent” within the meaning of the present invention is meant any hydrocarbon compound used alone or in combination to dissolve one or more products.

The sample can be chosen from raw organic extracts and biological liquids.

Said process can take place with a molar ratio (molecules to be analyzed) / (matrix molecules) of between 1: 33333 (PFPT3P) and 1: 105715 (CHCA), for all the analytes used (except angiotensin II). The concentration of the matrix solution used is always 10 mg / ml.

The present invention also relates to a method of characterization and / or quantification of molecules, advantageously having a molecular mass less than 800 Da, present in a sample, by time-of-flight mass spectrometry with desorption / ionization under laser assisted by matrix or MALDI-TOF-MS comprising the steps of:

a) Manufacture of a matrix with the PFPT3P compound of formula:

PFPT3P

Exact mass: 462.96 according to the process of the invention as defined above;

b) Mixing said sample to be analyzed with said matrix, optionally in the presence of an organic solvent or water;

c) Vaporization of the solvent or water, if applicable;

d) Co-crystallization of said matrix and of said molecules and formation of a matrix crystal comprising said molecules distributed throughout said crystal;

e) Ionization by a laser beam of said matrix co-crystallized with the sample, obtained previously;

f) Establishment of a spectrogram.

Brief Description of the Figures

Figure 1 shows the MALDI-TOF mass spectra of two commercially used matrices, HCCA and DHB, tested without analytes.

FIG. 2 represents the molecular structure of the matrices tested.

FIG. 3 represents the structure of all the compounds analyzed (except in the case of the complex peptides which are angiotensin II and gramicidin).

FIG. 4 represents a MALDI spectrum of PFPT3P without analyte (A).

Colchicine (B) and sparteine (C) spectra obtained for simple compounds using PFPT3P as a matrix. (D): DCM extract spectra of C. autumnale showing colchicine and one or more of the isomers 2- or 3demethyldemecolcine (I), N-deacetyl-N-formyl-colchicine, 2- or 325 demethylcolchicine (II). Spectra of RYTMOPASC® solution (E) and plasma enriched in strychnine (F) using PFPT3P as a matrix. * signals associated with the matrix.

FIG. 5 represents the structures of the minor alkaloids detected in the extract of C. autumnale (A, B and C) and in the RYTMOPASC® solution (D).

EXAMPLES

Example 1: Materials and Methods

1.1. Chemicals and reagents

Methanol (MeOH), dichloromethane (DCM), acetonitrile (ACN) and quality water for high performance liquid chromatography (HPLC) were obtained from Carlo Erba reagents (Val-de-Reuil, France). Colchicine, coumarin, pilocarpine, dexamethasone-21-acetate, gramicidin (AD, 1883.3 Da), standard mixture of HPLC peptides, trifluoroacetic acid (TFA) and CHCA were obtained from Sigma-Aldrich (Saint-Quentin fallavie, France ). The standard mixture of HPLC peptides contains approximately 0.5 mg of each of the following constituents: angiotensin II (1046.2 Da), leu-enkephalin (555.6 Da), Met-enkephalin (573.7 Da), Gly-Tyr (238.2 Da) and Val-Tyr-Val (379.5 Da). Spartein and harmine have been isolated during various research projects conducted at the SONAS Institute. Quercetin was obtained from Extrasynthèse (Genay, France), DHB from Avovado research Chemicals / Alfa Aesar (Karlsruche, Germany), curcumin from Fluka / Sigma-Aldrich and strychnine from ProlaboA / WR (Saint-Quentin Fallavier, France). Human plasma was generously provided by Nathalie Clément (UFR Santé, Department of Pharmacy, University of Angers, France).

1.2. Synthesis of bithiophenic matrices

For practical reasons, abbreviations have been used for all of the matrices discussed including the CHCA and DHB matrices (Figure 2), and the systemic names and formulas are provided in Table 1 below.

Table 1: Bithiophenic matrices evaluated for their matrix properties

Last name Abbreviation Structure Formula 2a 3- (5'-Methylsulfanyl [2,2 '] bithiophenyl-5y Isu Ifany l) itrile propion MT ₃ P JEXj) NC ^ _S ^ ^SS _s / C12H11NS4 2b 3- (5‘- Cyanomethylsulfanyl- [2,2 '] bithiophenyl-5- ylsulfanyl) propionitrile CMT3P _r -S. q ί 1 — SC S. NC [P © - / V C13H10N2S4 2c 3- (5'-Ethylsulfanyl- (2,2 '] bithiophenyl-5- ylsulfanyl) propionitrile ETsP ^ ^cn C ₁₃ H ₁₃ NS4 2d 3- (5 '- / so-propylsulfanyl- [2,2 '] bithiophenyl-5- ylsulfanyl) propionitrile IPT3P T ty-V C14H15NS4 2nd 3- (5'- Pentafiuorophenylmethy Isulfanyl- [2,2 '] bithiophenyl-5- ylsulfanyl) propionitrile PFPTsP T XH © Ci ₈ H ₁₀ F ₅ NS ₄ 3 [2,2 '] BithiophenyI5-carboxylic acid BTA 0 i-S / S-z ^ OH C9H6O2S2

The MT3P matrix was synthesized according to a protocol previously described [20]. All new bithiophenic matrices (except for BTA) were created following a general protocol. All the reactions were carried out under an inert nitrogen atmosphere. All chemicals were used as received without further purification. The division diagrams have been designated as follows: s (singlet), d (doublet), t (triplet), m (multiplet). Compound 2a was synthesized according to [20]. The other compounds were prepared by following the same

1- CsOH / MeOH -►

2- R-X (X = Br, I)

2a-e

3- (5'-Cyanomethylsulfanyl- [2,2 '] bithiophenyl-5-ylsulfanyl) propionîtnle CMT3P (2b):

3- [5 ^, - (2-Cyano-ethylsulfanyl) - [2,2 '] bithiophenyl-5-ylsulfanyl] propionitrile (1) (110 mg, 0.33 mmol, 1 equiv.) Was dissolved in dry DMF ( 8 ml) and the solution was smoked with nitrogen for 45 minutes. Then an oxygen-free methanolic solution (5 ml) of cesium hydroxide (63 mg, 0.41 mmol, 1 equiv.) Was added and the mixture was stirred at room temperature for 1 hour. Finally, bromoacetonitrile (50 mg, 0.42 mmol, 1 equiv.) Was added, and the solution was stirred for a further 3 hours at room temperature. The solvent was evaporated to dryness under vacuum at reduced pressures, and the residue was purified by isocratic CC (hexane / DCM: 1/1) to give the compound 2b as a brown solid (75 mg, 70, 7%).

¹ H NMR (CDCb) δ 7.32 - 7.30 (m, 1H), 7.14 - 7.07 (m, 3H), 3.50 (s, 2H), 3.01 (t, 2H), 2.65 (t, 2H). ¹³ C NMR (CDCb) δ 143.0, 141.4, 138.3, 136.8, 131.4, 128.8, 125.3, 125.0, 117.8, 116.2, 33.8, 24.9, 18.4. HRMS (MALDI) calculated for C ₁₃ HioN ₂ S ₄ ([M] ⁺ ) 321.9727, 321.9626 found.

Compounds 2c, 2d and 2e were prepared following the procedure for compound 2b. The halides used for the three reactions were, respectively, diethyl sulphate (3.5 equiv.), 2-bromopropane (3 equiv.) And a-bromo2,3,4,5,6-pentafluorotoluene (1 , 5 equiv.):

3- (5'-Ethylsulfanyl- [2,2 '] bithiophenyl-5-ylsulfanyl) propionitrile ET3P (2c):

Yield 90%, yellow solid. ¹ H NMR (CDCb): δ 7.12 (d, J = 3.7 Hz, 1 H), 7.03 7.00 (m, 3H), 2.99 (t, J = 7.2 Hz, 2H), 2.84 (q, J = 7.3 Hz, 2H), 2.65 (t, J = 7.2 Hz, 2H), 1.30 (t, J = 7.3 Hz, 3H). ¹³ C NMR (CDCb): δ 142.4, 139.5, 136.9, 135.0, 134. 4, 130.0, 124.6, 124.2, 117.9, 33.9, 33.0, 18.4, 14.9. HRMS (MALDI) calculated for C13H13NS4 ([M] ⁺ ) 310.9930, 310.9756 found.

3- (5'-lsopropylsulfanyl- [2,2] bithiophenyl-5-ylsulfanyl) -propionitrile IPT3P (2d): Yield 62%, yellow solid. ¹ H NMR (CDCb) δ 7.13-7.11 (m, 1H), 7.06 - 7.01 (m, 3H), 3.24 - 3.11 (m, 1 H), 3.03 - 2.95 (m, 2H), 2.65 (td, J = 7.1, 2.2 Hz, 2H), 1.30 (d, J = 6.7 Hz, 6H). ¹³ C NMR (CDCb) δ 142.4, 140.3, 136.9, 136.0, 133.7,

130.1, 124.6, 124.3, 117.9, 42.0, 33.9, 23.1, 18.4. HRMS (MALDI) calculated for C14H15NS4 ([M] ⁺ ) 325.0087, 324.9964 found.

3- (5'-Pentafluorophenylmethylsulfanyl- [2,2 '] bithiophenyl-5ylsulfanyl) propionitrile PFPT3P (2e): Yield 74%, bright green-yellow solid. ¹ H NMR (CDCb) δ 7.09 (dd, J = 37.3, 3.7 Hz, 2H), 6.96 (dd, J = 31.1.3.7 Hz, 2H), 4.00 (s, J = 8.1 Hz, 2H), 3.00 (t , J = 7.2 Hz, 2H), 2.66 (t, J = 7.2 Hz, 2H). ¹³ C NMR (CDCb) δ 142.1, 141.8, 137.1, 136.8, 131.3, 130.9, 124.8, 124.7, 117.8, 33.9, 18.4.HRMS (MALDI) calculated for C ₁₃ H ₁₀ N ₂ S4 [M] ⁺ 462.9616, 462.9541 found .

For the acid matrix [2,2 '] Bithiophenyl-5-carboxylic BTA (3): the compound was synthesized by oxidation of 2,2’-bithiophenyl-5-carbaldehyde using the Tollens reagent [21],

The structure of all the new compounds was confirmed by NMR spectroscopy as well as by high resolution mass spectrometry (MS).

1.3. Instruments

All experiments by MALDI were carried out in linear positive mode on a time-of-flight mass spectrometer (TOF) Bruker Biflex III (Bruker Daltonics, Bremen, Germany) equipped with a pulsed nitrogen laser, 337 nm (VSL- model 337Î, Laser Sciences Inc., Boston, Massachusetts). All of the simple compounds were tested at a laser energy of 35% (60.55 μ J), which was 10% (27.3 pJ) above the ionization threshold of bitiophenic matrices. Complex mixtures such as DCM extract from C. automnale, stychnine-enriched serum and RYTMOPASC® solution were tested at 40% laser energy (67.2 μ J). Spectra were acquired in a mass range of 20-2000 m / z. The acceleration voltage was set at 19 kV, the pulse ion extraction was 200 ns, and the laser frequency was 5 ns. In order to exclude the effects of laser desorption / ionization (LDI), all samples were also analyzed without the matrices under the same conditions and no signal was observed.

The UV-VIS absorption profiles (200-430 nm) of all the matrices were obtained using the LAMBDA 950 UVA / is / NIR spectrophotometer (PerkinElmer, Walthamn, USA) and the NMR experiments were carried out on a JEOL spectrometer. ECZ 400 MHz (Tokyo, Japan). The high resolution mass spectra were acquired on a SpiralTOF ™ MALDI TOF / TOF mass spectrometer (JEOL, Tokyo, Japan).

1.4. Preparation of matrix solutions

Except for the CHCA matrix, all matrix solutions were prepared at a concentration of 10 mg / ml. The bithiophenic matrices were dissolved in DCM and the DHB matrix was dissolved in 70% ACN (aq) containing 0.1% TFA. A-Cyano-4-hydroxycinnamic acid was dissolved until saturation in 70% ACN (aq) containing 0.1% TFA and then diluted in 30% ACN (aq) containing 0 , 1% TFA at a ratio of 1: 2. Two equivalents of each matrix solution were mixed with one equivalent of the standard working solution, giving final matrix concentrations of 22.5 mM for MT3P, 14.4 mM for PFPT3P, 21.4 mM for ET3P, 20, 7 mM for CMT3P, 31.7 mM for BTA, 20.5 mM for IPT3P, 43.3 mM for DHB and 45.81 mM for CHCA.

1.5. Basic solutions, working solutions and sample deposit

The basic solutions of each simple compound were prepared in MeOH at a concentration of 2.6 μΜ and stored at -20 ° C until use. For the standard mixture of HPLC peptides: 0.5 mg of the lyophilized precipitate was dissolved in 500 μl of MeOH (aq).

The working solutions were freshly prepared for each experiment by diluting the base solutions with MeOH at 70% (aq) (0.1% TFA) at a 1: 2 ratio. Subsequently the working solutions were mixed with the matrix solutions at a ratio of 1: 2 and 0.7 μl of the mixture were optionally deposited on a stainless steel plate for MALDI. The final concentration of the sample was 200 nmol per sample point. The samples were deposited in four copies. The RYTMOPASC® solution was diluted 1: 1 with MeOH and mixed with the matrix solution at a ratio of 1: 2.

1.6. Extraction and treatment of Colchicum autumnale seeds

The crushed seeds of C. autumnale were basified using 25% ammonia (aq) before being extracted with DCM for 4 hours using a Soxhlet device. The excess solvent was removed under reduced pressure by a rotary evaporator. The working solution of C. autumnale was prepared in DCM at a concentration of 1.2 mg / ml.

1.7. Preparation of strychnine-enriched plasma samples

The strychnine was dissolved in MeOH giving a concentration of 0.72 mg / ml. 10 µl of this solution was mixed with 900 µl of plasma to give a 0.18 mM working solution. The latter was then mixed with the CHCA or PFPT3P matrix solution at a ratio of 1: 2. In order to evaluate the ionization properties at lower concentrations, the working solution was further diluted with the plasma, giving a final concentration of 2.1 μΜ. In order to facilitate sufficient miscibility with the aqueous working solutions, the PFPT3P matrix solution was exceptionally prepared in a mixture of MeOH and DCM for this experiment.

1.8. Data collection and processing

All the experiments were carried out in automatic mode in order to facilitate an impartial comparison of the signal intensities acquired. The samples were deposited in four copies. The spectra were acquired from 30 uniformly distributed locations, each irradiated by 5 laser shots. A total of 150 acquisitions were added for each of the four copies. The experiments were carried out on three different days and the results were averaged. In order to assess the impact of the formation of matrix noise, the matrix suppression effect score (MSE) was calculated for samples selected according to a previously published protocol [13]. In summary, all the signals of the analyte have been divided by the sum total of all the signals (analyte + matrix). Weak signals (less than 5% of the base peak) were not taken into account.

X signals from the analyte

MSE score = ¹

X analyte signals + X matrix signals The equation gives a factor ranging from 0 to 1, providing a good indication of the quality of the spectrum. Values close to 1 indicate low matrix noise, those close to 0 indicate strong matrix noise.

Example 2: Results

Selective MALDI matrices can provide a useful tool for the analysis of complex mixtures and the MT3P matrix has been previously reported as a model compound for the selective ionization of alkaloids [19].

The present invention therefore focused on the development of new bithiophenic matrices and analyzed their specific ionization profiles. One of the most basic requirements of a good matrix is its absorption at the emission wavelength of the MALDI laser. The structural modification of the side chains attached to the bithiophenic nucleus had little impact on the absorption profile. At 50 µM in DCM, the optical densities range between 0.750 and 1.036 while the molecular extinction coefficients remain in the range 11561-21558, as described in Table 2 below.

Table 2 molar extinction coefficients for the bithiophenic matrices determined at 337 nm in DCM at a concentration of 50 μΜ. The two matrices of CHCA and DHB were not soluble in DCM at the given concentration and the band values have been found from the literature [22]

Matrix S (L.moM.cm ' ¹ ) at 337 nm BTA 18000 ± 3600 PFPT3P 20400 ± 4100 ET3P 15600 ± 3100 IPT3P 20000 ± 4000 CMT3P 21600 ± 4300 CHCA 17800 ± 3600 DHB 3340 ± 670

Remarkably, of all the matrices tested, the DHB matrix showed the lowest absorption and required the highest laser energy (> 50%,

80.5 pJ) to be ionized. All of the matrices showed excellent ionization at 25% laser energy (47.25 pJ) and therefore represented the most interesting matrix candidates. Subsequently, the bithiophenic matrices were tested against pure analytes and evaluated for the degree of formation of matrix noise. Based on these results, the selected matrices were then tested on highly complex samples. These results are discussed below.

2.1. Ionization of single compounds and formation of matrix noise All the bithiophenic matrices were tested on pure compounds belonging to different chemical groups comprising five alkaloids chosen for their pharmacological importance. The structures of all test compounds (except for angiotensin II and gramicidin, two complex peptides) were shown in Figure 3. This strategy served two purposes. First, the yields of the ions obtained for the alkaloids were directly compared allowing a rough estimate of the individual ionization power of the matrices. Second, and more importantly, matrix selectivity has been assessed on compounds other than alkaloids covering a wide range of chemical groups.

The results of these experiments are described in Table 3 below.

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It has been found that, except for BTA, virtually all bithiophenic matrices have shown a clear preference for ionization over alkaloids. Α-Cyano-4-hydroxycinnamic acid gave the strongest signals for all the alkaloids but also showed significant ionization of several compounds outside this group. Interestingly BTA presented an ionization profile similar to that of the CHCA matrix but with generally lower signal intensities. It should be noted that the matrices of BTA and CHCA have a COOH function, which is not the case for the rest of the bithiophenic matrices. Therefore, it is reasonable to assume that the presence of COOH generally facilitates a broader and less selective ionization profile. 2,5-Dihydroxybenzoic acid, which also contains a COOH function, however did not ionize any of the test compounds at the given ionization energy.

Except for BTA, all bithiophenic matrices are equipped with a 2-cyanoethylthio function attached to the bithiophenic cyclic system, which was previously reported as essential for the matrix effect of MT3P [19], Furthermore, it has been observed that replacing the terminal CN function with OH or NH2 led to a total loss of the matrix properties (data not shown).

Among all the bithiophenic matrices, PFPT3P exhibited the strongest ionization of the alkaloids and was therefore chosen for subsequent experiments.

As shown in Table 4 below, the overall signal intensities of PFPT3P were slightly lower than those obtained for CHCA but the MSE scores appeared to be of the same magnitude. While sparteine performed best when analyzed by PFPT3P (MSE score 0.493 vs. 0.177), CHCA appears to have produced better spectrum qualities for harmonic (MSE score 0.773 vs. 0.369 for PFPT3P). However, this result should be interpreted with caution. Indeed, current experiments and a previous study [23] identified [M + Na +] at 212.0 m / z as a strong and important matrix signal of CHCA. However, the harmony has a monoisotropic mass of 212.1 Da, so that a superposition of the signals of the matrix and of the analyte can be envisaged.

Compared to CHCA, the bithiophenic matrices exhibited fewer matrix signals, thereby minimizing the risk of superimposing the matrix signal and the analyte signal. The matrix ions of the bithiophenic matrices come mainly from their molecular and quasi-molecular ions as well as from the sulfur-carbon cleavage in position 2 or 5 ’. For PFPT3P, these signals appear at 282.8, 411.0 and 463.0 m / z. The identification of specific matrix ions can possibly optimize the choice of matrix relative to the mass of the analyte.

Table 4: MSE scores calculated for CHCA and PFPT3P. ‘Overlay of matrix signals and analyte

Alkaloid CHCA PFPT3P Colchicine 0.445 ± 0.063 0.558 ± 0.046 Harmine 0.773 ± 0.211 * 0.369 ± 0.023 Pilocarpine 0.187 ± 0.017 0.173 ± 0.039 Sparteine 0.177 ± 0.021 0.493 + 0.046 Strychnine 0.225 ± 0.053 0.385 ± 0.024

In order to exclude direct LDI as a factor contributing to the observed signal intensities of the analyte, all the compounds were additionally tested without a matrix at 35% laser energy (60.55 pJ) where no ionization was observed. Curcumin, the polyphenol of Turmeric also described as a potential matrix, showed self-ionization from a laser energy of 45% (73.85 pJ) [24], These inherent matrix properties can also explain the signal of Fairly intense molecular ion observed for most bithiophenic matrices including PFPT3P. Similar observations were obtained for quercetin, which showed self-ionization at 50% laser energy (80.5 pJ).

2.2. Analysis of complex mixtures

As previously reported, CHCA gave the strongest signal intensities for the pure compounds tested followed by PFPT3P, which facilitated selective ionization of the alkaloids. Being of little relevance during the analysis of simple compounds, selectivity becomes essential when it is a question of mixtures with several components. For this reason, the two matrices were then tested on complex mixtures, i.e. a crude extract of C. autumnale, the RYTMOPASC® solution, and plasma enriched in strychnine.

2.2.1. Crude extract of Colchicum autumnale

C. autumnale (Colchicaceae), commonly known as the crocus or fall crocus, wild saffron or naked lady, is a poisonous plant containing colchicine as one of its main constituents. The alkaloid is commonly used as a gout medicine but is also known to have serious toxic effects [25]. Lethal poisoning by accidental ingestion of C. autumnale is sometimes reported [25, 26] because the plant is often confused with edible wild garlic (Allium ursinum, Amaryllidaceae) [25]. In the zootechnical field, C. autumnale is also a dangerous contaminant of hay causing serious to fatal poisoning of cattle and horses in Europe [27],

To assess the ionization properties of CHCA and PFPT3P vis-à-vis complex mixtures, a DCM extract obtained from seeds of C. autumnale was analyzed. Similar to the results obtained for single compounds, CHCA gave the strongest signal intensities for colchicine, 102.9 (± 12.7) vs 24.7 (± 2.0). With an average yield of 0.72-0.75%, the latter is, as previously mentioned, known as a major constituent of the seeds of the plant [28]. Besides colchicine, minor alkaloids such as 2- or 3-demethyldemecolcine, [MH] ⁺ at 356.2 m / z, as well as one or more of the three isomers reported - namely N-deacetyl- N-formyl-colchicine and 2- or 3demethylcolchicine, [M + H] ⁺ at 386.2 m / z - were detected in the extract by CHCA and PFPT3P (Figure 4D, Figure 5A, B, C) [29 ].

The overall results indicate that CHCA and PFPT3P represent suitable matrices for the analysis of C. autumnale. CHCA gave stronger signals for colchicine but no significant difference between CHCA and PFPT3P was observed for the rest of the tropolone alkaloids detected.

2.2.2. Analysis of the RYTMOPASC® solution

After examining the extract of C. autumnale, an experiment was carried out on the RYTMOPASC® solution, a homeopathic preparation based on plant tinctures containing in particular 9.5% of an ethanolic extract of Cytisus scoparius (Fabaceae). The latter is a known source of sparteine, an alkaloid with antiarrhythmic properties but also toxic effects [30, 31]. Despite the induction of a strong ionization of this alkaloid during tests carried out on purified compounds, CHCA did not ionize sparteine in the RYTMOPASC® solution, while its quasimolecular ion [M + H] ⁺ was clearly detected using PFPT3P as a matrix (Figure 4E). In addition to C. scoparius, the RYTMOPASC® solution also contains 1% of an ethanolic extract of virid Veratrum (Melanthiaceae) (dilution D2 at 1: 100) and a signal indicating the presence of rubijervine (Figure 5D) ([M + H] ⁺ ) at 414.4 m / z was detected in the spectrum. Veratrum viride is a recognized source of rubijervine [32], a terpene alkaloid of the solanidine type known to have high cardiac toxicity [33]. Although no specific quantification has been carried out, these results are fairly encouraging in that homeopathics generally contain relatively small amounts of bioactive compounds. In this case the raw extract of virid Veratrum was diluted by a factor of 1: 10,000. It also suggests that some homeopathics, by definition associated with very high dilutions of active ingredients, may still contain significant amounts of bioactive compounds. Also, in the interest of consumers, safety regulations comparable to those implemented for phytopharmaceuticals and synthetic drugs should also be applied for these products.

As previously discussed, sparteine and colchicine showed close signal intensities when analyzed by CHCA as simple compounds. While the latter was also easily detected in the raw extract of C. autumnale, the sparteine could not be ionized in the RYTMOPASC® solution. This observation is more reasonably explained by the higher complexity of the sample. Next to Spartium scoparium (9.5%), the RYTMOPASC® solution also contains extracts of Crategus species (38.7%), Lilium lancifolium (24%) and Apocynum cannabinum (9.5%), just to mention those appearing in the highest quantities. Under these conditions PFPT3P presented significant advantages compared to CHCA, an observation which was also confirmed by the analysis of plasma enriched in strychnine.

2.2.3. Plasma enriched in strychnine

Strychnine is a pharmacologically active and toxic alkaloid, which is also listed as a doping agent by the World Anti-Doping Agency (WADA). It represents a known contaminant in food supplements, so its detection in complex mixtures is an important factor for the quality and control of doping [34], As for many alkaloids, the latter is mainly carried out by LC methods or GC-MS, which generally require one or more stages of pre-purification of the sample. Especially when processing blood samples, these methods (deproteinization, etc.) take a long time.

To assess the matrix properties of CHCA and PFPT3P, a simplified one-step MALDI protocol was applied to the analysis of plasma enriched in strychnine (cf. 1.7.). In summary: the plasma enriched in strychnine was directly mixed with the matrix solutions of CHCA and PFPT3P before being deposited on a sample support.

Similar to the observations made for sparteine in the RYTMOPASC® solution, strychnine was not ionized by CHCA, but gave a strong signal in the PFPT3P spectrum (Figure 4F) at a concentration of 0.18 mM. By further diluting the working solution, the signal was always detectable even at the lowest of the tested concentrations of 2.1 μΜ (7.4 pg / ml). The latter remains higher than the linearity range of LC-MS techniques (0.5500 ng / ml) [35] but the simplicity of the method makes it an interesting tool for the rapid screening of complex mixtures. In addition, it is reasonable to assume that the detection limit (LOD) of strychnine in human plasma is less than

2.1 μΜ and that an optimized MALDI protocol could further improve the sensitivity of PFPT3P.

In conclusion, the current overall results confirm the excellent selectivity of PFPT3P towards alkaloids as well as its advantages compared to CHCA for the analysis of complex samples.

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Claims (17)

1. Use of the PFPT3P compound of formula (I): PFPT3P Exact mass: 462.96 as matrix in a desorption / ionization mass spectrometry device under laser assisted by matrix or MALDI. 2. Use according to claim 1, wherein said MALDI device is coupled to a time-of-flight analyzer. 3. Use according to any one of claims 1 or 2, for qualitatively and quantitatively analyzing a mixture of molecules comprising at least one molecule of mass <800 Da. 4. Use according to claim 3, wherein said molecule of mass <800 Da is chosen from alkaloids. 5. Use according to claim 4, in which the alkaloids are chosen from: • The group of Azolidines; • The Azines group; • The Tropanes group; • The Quinoline group; • The group of Isoquinolines; • The group of Indoles; • The Terpenoids group; • The Betaine group; • The Pyrazoles group. 6. Use according to claim 4, in which the alkaloids are chosen from (i) Aniracetam, Anisomycin, CX614, Dextromoramide, Diphenyl prolinol, Domic acid, Histapyrrodine, Kainic acid, Methdilazine , Oxaceprol, Prolintane, Pyrrobutamine, Hygrine, Cuscohygrine; (ii) Piperidine, Conicine, Trigonelline, Acrecaidine, Guvacine, Pilocarpine, Cytisine, Nicotine, Sparteine, Pelletierine; (iii), atropine, L-Hyoscyamine, Cocaine, Ecgonine, Scopolamine; (iv) Acridine, Bicinchoninic Acid, Broxyquinoline, Chlorquinaldol, Cinchophen, Clioquinol, Dequalinium, Dihydroquinine, Dihydroquinidine, Hydroxychloroquine, 8-Hydroxyquinoline, Iodoquinol, Kynurenic Acid , Mefloquine, Nitroxoline, Oxycinchophen, Primaquine, Quinine, Quinidine, TSQ, Topotecan, Xanthurenic Acid, Strychnine, Brucine, Veratrine, Cevadine, Echinopsin, Aminoquinolines such as la Chloroquine, Hydroxychloroquine, Primaquine, 8-Aminoquinolines such as Tafenoquine, Rhodoquine, Pamaquine; (v) Dimethisoquine, Quinapril, Quinapirilat, Debrisoquine, 2,2'-Hexadecaméthylènediisoquinolinium dichloride, N-Laurylisoquinolinium bromide, Narcéine, Hydrastine, Berberine, natural opium alkaloids such as la Morphine, Codeine, Thebaine, Papaverine, Narcotine, Noscapine, semisynthetic opium alkaloids such as Hydromorphone, Hydrocodone, Heroin, synthetic opium alkaloids such as Fentanyl, Pethidine, Methadone, Propoxyphene; (vi) ergolines such as ergot alkaloids such as ergometrine, erggotamine, ergosine, ergovaline, ergokryptine, ergocornine, ergocristine, lysergic acid, Ergine, LSD, (vii) Beta-carbolines such as Harmine, Yohimbine, Reserpine, Emetine; (viii) napel aconite alkaloids such as Aconitine, Solanidine, (ix) Solasodine, Batrachotoxine, Delphinine, (x) steroids such as Solanine, Samandarin; (xi) Muscarine, Choline, Neurine. 7. Use according to claim 3, wherein said mixture is a mixture chosen from crude organic extracts, or any fraction obtained from said crude organic extracts, or biological liquids, of human or animal origin. 8. Use according to claim 7, in which the biological fluid is chosen from blood and plasma, the fluids of the genital mucosa, the fluids of the skin, the fluids of the integuments, the effusion liquids and closed cavities, the liquids of the digestive and urinary system. 9. Compound PFPT3P of formula: PFPT3P Exact mass: 462.96 10. Use of a compound according to claim 9 for manufacturing a matrix intended to be used in a desorption / ionization mass spectrometry device under matrix-assisted laser or MALDI. 11. Method for manufacturing a matrix intended to be used in a laser-assisted desorption / ionization mass spectrometry device A matrix or MALDI consisting in crystallizing a compound according to claim 9. 12. The method of claim 11, wherein said MALDI device is a mass spectrometer coupling a laser ionization source assisted by a matrix and a time-of-flight analyzer. 13. Method according to any one of claims 11 or 12, characterized in that said crystallized compound according to claim 9 is then dissolved in an organic solvent or water comprising a sample to be analyzed, said solvent or water being then vaporized ultimately forming a crystallized matrix of compound according to claim 9 comprising the sample to be analyzed. 14. The method of claim 13, wherein the sample is chosen from crude organic extracts and biological liquids. 15. Method according to any one of claims 13 or 14, in which the molar ratio (molecules to be analyzed) / (matrix molecules) is from 1: 33333 to 1: 105715. 16. Method for characterizing and / or quantifying molecules, advantageously having a mass <800 Da, present in a sample by time-of-flight desorption / ionization mass spectrometry under matrix assisted laser comprising the steps of: a) Manufacture of a matrix with a compound according to claim 9; b) Mixing said sample with said matrix, optionally in the presence of an organic solvent or water; c) Vaporization of the solvent or water, if applicable; d) Co-crystallization of said matrix and of said molecules and formation of a matrix crystal comprising said molecules distributed throughout said crystal; e) Ionization by a laser beam of said matrix co-crystallized with the sample, obtained previously; f) Establishment of a spectrogram. 17. Method according to claim 16, characterized in that the sample is a biological liquid chosen from blood, plasma, liquids of the genital mucous membranes, liquids of the skin, liquids of the integuments, effusion liquids or closed cavities, liquids from the digestive or urinary system. 1/4 Irrtens.